MADCAM1

GENE POLYMORPHISMS AND THE OUTCOME OF THE ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION

Z. Ambruzova 1 , F. Mrazek 1 , L. Raida 2 , A. Stahelova 1 , E. Faber 2 , K. Indrak 2 , M. Petrek 1 1 Laboratory of Immunogenomics and Proteomics, Department of Immunology, 2 Department of Haematooncology Palacky University and University Hospital Olomouc, Czech Republic

Supported by Czech Govt. Funding MSM 6198959205 and IGA MZ CR NR 9099

INTRODUCTION

 various outcome

non-HLA gene polymorphisms

are intensively studied for their possible relevance for allogeneic haematopoietic stem cell transplantation (aHSCT) 

migration and distribution of activated donor T cells

to the recipient mucosal sites and parenchymal target organs is important for development of graft-versus-host disease (GVHD)  organ specific „homing“ of donor cells is mediated via interaction between

adhesion molecules

and their ligands

MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1

(

MAdCAM-1)

 glycoprotein expressed on the surface of the endothelial cells (high level of expression in Peyer ´s patches and mesenteric lymph nodes) 

ligands

: α4β7 integrins (LPAM-1, CD49d/β7) L-selectin (CD62L) 

function

: lymphocyte traffic to mucosal sites Eksteen et al, Clin Med 2004; 4:173-80 adhesion of leucocytes on the endothelium aHSCT donor T cells „homing“ into the recipient mucosal tissue

MADCAM1

GENE POLYMORPHISM

 many single nucleotide polymorphisms (SNPs) were described throughout the

MADCAM1

gene (chromosome 19)  particular variants of the

MADCAM1

gene may affect structure and/or expression of MAdCAM-1 molecule  SNPs selected to our study: rs 758502 T/C promotor rs 2302217 G/A exon 3 synonymous (Pro/Pro) rs 3745925 G/T exon 4 non-synonymous (His/Pro)

AIM OF THE STUDY

 to investigate whether the selected are the risk factors for development of the serious complications after aHSCT

MADCAM1

gene SNPs

MADCAM1 gene SNPs

rs 758502 T/C rs 2302217 G/A rs 3745925 G/T acute or chronic GVHD overall survival

INVESTIGATED SUBJECTS

aHSCT pairs Age – median (range)

Patients Donors

Recipient gender

Female Male

Diagnosis

Acute leukaemia (AML, ALL) Chronic leukaemia (CML, CLL) Non-Hodgkin lymphoma Other

Conditioning regimen

Non-myeloablative Myeloablative

Donor HLA compatibility

Identical Mismatched

87

44 (18-61) 40 (18-69) 37 50 43 15 14 15 48 39 87 0

Donor type

Related Unrelated

Cell source

PBSC Bone Marrow

Acute GVHD

Grade 0-I Grade II Grade III Grade IV 70 17 86 1 53 23 4 8

Chronic GVHD

None 56 Limited 17 Extensive 14

METHODS

1.

Genotyping

Polymerase Chain Reaction with Sequence Specific Primers (PCR SSP) Primers designed according to the reference

MADCAM1

gene sequence: rs 758502 T/C rs 2302217 G/A rs 3745925 G/T

TT TC CC

MADCAM1

genotyping (rs758502)

2. Statistics

Conformity to the Hardy-Weinberg equilibrium: Chi-square test Differences between allele and genotype frequencies: Pearson logistic regression model Overall survival analysis: Kaplan-Meier, log-rank test and Cox regression analysis (SPSS software, version 15.0) ´s Chi squared test, Bonferroni correction for multiple comparisons, binary

MADCAM1 SNPs GENOTYPE FREQUENCIES IN PATIENTS AND DONORS

Patients

n = 87

Donors

n = 85

MADCAM1 T/C (rs758502)

Genotype CC Genotype TC Genotype TT

MADCAM1 G/A (rs2302217)

Genotype AA Genotype GA Genotype GG

MADCAM1 G/T (rs3745925)

0.43 (37) 0.47 (41) 0.10 (9) 0.26 (22) 0.51 (43) 0.24 (20) 0.53 (44) 0.37 (31) 0.10 (8) 0.35 (29) 0.40 (33) 0.24 (20) Genotype GG Genotype GT 0.64 (54) 0.33 (28) 0.77 (63)* 0.18 (15) Genotype TT 0.04 (3) 0.05 (4) ______________________________________________________________________ *

MADCAM1

(rs3745925) GG genotype frequency: p = 0.03, p corr >0.05 for comparison patients with donors

FREQUENCIES OF MADCAM1 IN PATIENTS WITH AND WITHOUT ACUTE GVHD GENOTYPES

MADCAM1 T/C (rs758502)

Genotype CC Genotype TC Genotype TT

Patients aGVHD+

n = 35 0.37 (13) 0.54 (19) 0.09 (3)

MADCAM1 G/A (rs2302217)

n = 34 Genotype AA Genotype GA Genotype GG 0.15 (5) 0.62 (21) 0.23 (8)

Patients aGVHD-

n = 49 0.45 (22) 0.43 (21) 0.12 (6)

p*

0.477

0.592

n = 48 0.31 (15) 0.46 (22) 0.086

0.23 (11) 0.948

MADCAM1 G/T (rs3745925)

n = 34 n = 48 Genotype GG Genotype GT 0.59 (20) 0.35 (12) 0.69 (33) 0.29 (14) 0.354

Genotype TT 0.06 (2) 0.02 (1) 0.367

___________________________________________________________________________ * comparison of particular homozygous genotype versus carriage of an alternative allele

FREQUENCY OF CHRONIC GVHD IN RECIPIENTS ACCORDING TO THEIR MADCAM1 rs2302217 GENOTYPES

p = 0.03

p corr = NS 70% 60% 50% 40% 30% 20% 10% 0% 64,7% 34,0% Genotype AA Genotype AG or GG

MULTIVARIATE ANALYSIS OF RISK FACTORS FOR OVERALL SURVIVAL AFTER aHSCT

Factor

aGVHD cGVHD

MADCAM1

rs2302217 AA genotype

p

0,00001 <0,00005 0,001

HR

4,35 5,7 2,99

Factors included to the analysis:

Diagnosis (malignancy vs. non-malignancy), type of the donor (related vs. unrelated), stem cell source, conditioning regimen, GVHD prophylaxis, donor-recipient gender combination (female donor in male recipient vs. others), aGVHD, cGVHD,

MADCAM1

gene variants

CONCLUSION

 preliminary data suggest that aHSCT

MADCAM1

rs2302217 AA genotype in recipients may be associated with the risk of chronic GVHD and decreased overall survival after  possible impact of

MADCAM1

SNP variants for aHSCT outcome has to be confirmed in substantially larger cohorts of donor-recipient aHSCT pairs

Dept. of Immunology, Medical Faculty Palacky University and University Hospital Olomouc

Prof. Martin Petrek Frantisek Mrazek Anna Stahelova Jana Onderkova Silva Zachova

Dept. of Haematooncology, Medical Faculty Palacky University and University Hospital Olomouc

Prof. Karel Indrak Ludek Raida Edgar Faber

Thank you for your attention…